CA1056817A - Penicillins - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A class of novel .beta.-amino penicillins of formula (IV) useful as antibacterial agents:

(IV) and non-toxic salts and hydrolysable esters thereof, in which formula R is a C1 to C6 straight or branched chain alkyl group, a C5 to C7 cycloalkyl or cyclo-alkenyl group; R1 is hydrogen or a C1 to C6 alkyl group; or R and R1 taken to-gether represent a C5 to C6 alkylene group which may be interrupted by a hetero atom. According to the process of preparation 6-aminopenicillanic acid or a salt or ester thereof or the reaction product of 6-aminopenicillanic acid with an organo silicon compound is treated with a reactive acylating derivative of an acid of the general formula (V)

Description

~5~7 This in~ention rel.ates to certain new penicillins and is parti-cularly concerned with a new class of penicillins which are derivatives of 6-aminopenicillanic acid and which are oE value as antibacterial agents, as nutritional supplements in animal food, as agents for the treatment of mastitis in cattle and as therapeutic agents in poultry and animals, including man, in the treatment especially of infectious diseases caused by Gram-positive and Gram-negative bacteria.
6-rD~x-aminophenylacetamido~ penicillanic acid (or ampicillin) has proved an extremely ~aluable antibacterial agent, having as it does a relatively broad spectrum of activity against both Gram-positive and Gram-negative bacteria. In view of the valuable properties of ampicillin, many attempts have ~een made to prepare new penicillins having structures closely related to ampicillin, in the hope that these novel structures will exhibit the same or superior properties. Thus, British Patent Specification 1,057,028 describes 6- ~-amino-~-phenylpropionamido) penicillanic acid ~hile our own British Patent Specification No. 1,133,448 describes structures of the type~

/ \ / 3 R.CH.CH .CO.NH - CH CH C - CH
¦ 2 l l 1 3 (I) NH2 CO N- CH.C02H

wherein R is a substituted phenyl group or heterocyclic group. British Patent Specification Nos. 960,896 and 962,943 describes inter alia penicillins of the formula (II):-ARYL - CH.CO.NH - CH - C~/ \C / CH

a2N 2 CO - N CH.CO2H (II) , , : , ,, , , , , , : , " . . ". .

G~

while Belgian Patent S~)ecification No. 641,516 describes compounds of formula (III) _ ~_ C _ CO NH CH CH \ / 3 IH2NH2 I I 1 3 (III) CO _ N CH CO2H
wherein the group R is alkyl, aryl or aralkyl.
Some of these known ~-aminopenicillins approach the antibacterial acti~ity of ampicillin _ vitro but there is no evidence that they are useful oral drugs. For example, ~etacin (the D-epimer of II; Aryl = phenyl) is said to give only poor oral blood levels in man (Current ~herap. Res., 1965, 7, 226).
This invention is based upon the discovery of a class of ~-amino penicillins which, upon oral administration, achieve higher and in some cases more prolonged blood levels than ampicillin. In general the new class of penicillins have a high level of activity against both Gram-positive and Gram-negative organisms, and thus many of these new compounds may offer therapeutic advantages over ampicillin.
According to the present invention there is provided a class of : ~ -amino penicillins of formula R C - CU2 CO - Nll CH CH C CH3 and non-toxic salts and hydrolysable esters thereof, in which :Eormula R is a C1 to C6 straight or branched chain alkyl group, a C5 to C7 cycloalkyl or cycloalkenyl group; R1 ls hydrogen or a Cl to C6 alkyl group; or R and R1 taken together represent a C5 alkylene grollp ~hich may be lnterrupted by a hetero atom, . .

,, . , ~ : .
: , :, , , :. , .

\

8~
The salts of the penicillins of ~ormula (I~) are non~toxic salts including non-toxic metallic salts such as sodium, potassium, calcium and aluminium, ammonium and substitu~ed ammonium salts, e.g.~salts of such non-toxic amines as trialkylamines (including triethylamine), procaine, dibenzylamine, N~benzyl-~-phenethylamine, l-ephenamine, N,N -dibenzylethylenediamine, dehydro-abietylamine, ~,N -bis-dehydro-abietyl-ethylenediamine, and other amines which have been used to form salts with benzylpenicillin. Acid addition salts of the compounds of formula (IV) are also included in the scope of this invention.
The esters of the penicillins of formula (IV) are non-toxic esters, particularly those known esters which are easily de-esterified in the body to give the parent penicillanic acids. ~xamples of such esters include acyloxyalkyl esters, particularly the acyloxymethyl esters such as acetoxy-methyl and pivaloyloxymethyl esters.
By way of example in penicillins of formula (IV) R may be methyl, ethyl, n- or _o- propyl, n~ sec- or tert- butyl, cyclopentyl, cyclohexyl, ` cycloheptyl, cyclohexenyl or cyclohexadienyl; ~1 may be hydrogen or a methyl or ethyl group. Alternatively R and Rl taken together ~ith the carbon atom to which they are attached may form a cyclopentyl, cyclohexyl or cycloheptyl ring which may be interrupted by an 0, S atom or a grou;p > NR2 ~here R2 represents hydrogen or a lower alkyl group such as methyl or ethyl.
Preliminary tests on representative members of the class of penicillins (IV) have shown the anticipated wide spectrum of activity against both Gram-positive and Gram-negative organisms. In general the compounds of this invention are antibacterially slightly less active in vit~ than ampicillin, but ,surprisingly high blood levels have been achieved on oral administration to small mammals.
The present ;lnvention further provides a process for the preparation of penicillins having the general formula (IV) and salts and esters ~a15~ 7 thereof in which 6-aminopenicillanic acid or a salt or ester thereof or the reaction product of 6-aminopenicillanic acid with an organo silicon compound is treated with a reactive derivative of an acid of the gèneral formula (V):-R 1.CH2COOH (V)X
where R and Rl are as hereinbefore defined and X is a nitrogen-containing group which may subsequently be converted into a primary amino grou~. The term X
particularly includes the so-called N-protected amino groups which can be found in the literature on peptide synthesis.
The conversion of the nitrogen-containing group X of the inter-mediate penicillin (VI) R / \ / CH3 R.C.CH2.CO.NH.CH - CH C - CH3 (VI) ` I
X CO - N - CH.COOH

to a primary amino group may be effected by hydrogenation or hydrolysis it being understood that both steps must be carried Otlt under conditions suffi-ciently mild that they do not distrupt the sensitive ~ lactam ri~g. ~hen the amlno group is protected by protonation only, conversion of X to Nnl2 merely requires adjustment of pH.
; The reactive derivative o~ the acid (V) may be the acid halide, azide anhydride, mixed anhydride, or the reactive intermediate formed from the acid and a carbodiimide or carbonyldiimidazole.
Examples of the nitrugen~containing group X which, in the inter-mediate penicillin (VI), can be con~erted into the primary amino groùp by a , '. ' ' . 4 _ . - : i ..... . : . , : .

: : , , .. , , , ~ ,. - , : ,, ~ ,,.. . : . .

.
` . ', , ' `, . . :` , : ' : ' ': ' ` ' :: , ::, , ~; : ~:: . . ., ~, . :

process of catalytic hydrogenation include the azido group, th~ benzyloxycarbonyl-amino group and substituted benzyloxycarbonylamino group.
Examples of the group X which may be converted into a primary amino group by a process of mild acid hydrolysis include enamine groups of general formula (VII), or tautomeric modifications thereof and o-hydroxyaryl-ideamino groups oE the general formula (VII~), or tautomeric modifications thereof. More detailed descriptions of the use of these groups in the synthesis of amino-penicilLins appear in British Patent Cipecifications 991,586 and 980,777:-R C N- ~ C N-R4~ ~ C H
~' \0/

(VII) (VIII) In structures (VII) and (VIII) the dotted lines represent hydrogen bonds. In structure (VII) R2 is a lower alkyl group of 1 to 6 carbon atoms, an aryl group or a lower alkoxy group of 1 to 6 carbon atoms. In structure (VII) R is hydrogen and R4 is Cl to C6 alkoxy. In structure (VIII) Z represents the residue of a substituted or urlsubstituted benzene or naphthalene ring.
In a still further process of the present invention the nitrogen-containing group X is a protonated amino group NH3. In such a process the reaction of the 6 aminopenicillanic acid and the reactive acid derivative is best carried out in weekly acid medium, the free primary amino group being , - , . . . . . . . .

,, . , , . . . . , :
~, , ~5~;83!~7 liberated at the end of the reaction by adjustment of the pll of the reaction' mixture from acid to neutral or mildly alkaline.
The novel penicillins (IV) are capable of existing in two epimeric forms and it is to be understood that the invention includes both the D- and L- forms as well as the DL- mixture. They are also capable of existing in the anhydrous form or as hydrates of various degrees of hydration.
Cert~in embodiments of the invention will now be illustrated by the :Eo:l,lowing specific Examples:- .
EX~MPLE I

Preparation of 6-LDL-3-amino-4-methyl pentanylamido~-penicillanic acid.
Isobutyraldehyde ~46 ml.), malonic acid (52 g) and ammonium acetate (77 g) were heated at reflux for 6 hours in 95% ethanol ~150 ml.).
The solution was allowed to cool, the crude product (25.5 g) collected by filtration and recrystallised from methanol-ether to give the pure 3-amino-4-methylpentanoic acid ~15.1 g. m.p. 194 ).
- The amino acid (6.5 g) wàs suspended in dry methylene dichloride (100 ml.) and dry hydrogen chloride gas passed for one hour. Thionyl chloride (7.6 ml.) was added to the suspension and gently refluxed fcr 4 hours. The resultant solution was clarified by filtration, evaporated to dryness under reduced pressure and the crude solid triturated with dry ether. The whi-te solid was collected by filtration~ quickly washed with dry et'her and dried in vacuo over phosphorus pentoxide to give the acid chloride hydrochloride,[(6.15 g)~
i.r.l-max (mull.) 1800 cm 1 acid chloride carbony.~ , shown by analysis and spectra to be contaminated with 4-5% of the amino acid hydrochloride, Solutions of the acid chloride hydrochlorlde (6.0 g) in methylene dichloride (100 ml.) and triethylamine ~2.9 &) in methylene dichloride (~5 ml.) were added concurrently over a period o~ 10 minutes to a solution of triethyl-ammonium 6-amino penicillanate (9.15 g) in methylene dichloride (75 ml.) at 5 .

.: ~
.
. .. .
:, . . ~. . .: , ~
: , - . , . - - , , : ., :

:

~fter stirring the reaction mixtu~e ~or one hour ~urther, the whlte precipitated solid was filtered, well washed with methylene dichloride and dried in vacuo over phosphorus pentoxide to yield 6~dl-3-amino-4-methyl pentanylamido] peni-cillanic acid ~(7.06 g) i.r. ~max (mull.) 1770 cm 1 (~ lactam C0, 1660 cm amide carbony1 . This material~ when subjected to paper chromatography in butanol:ethanol:water revealed a single ~one of inhibition at an Rf value of 0.19 and was estimated by colorimetric assay with hydroxylamine to be 59% pure.
XAMPLE II
Preparation of 6- C(d-amino-cyclollexyl) acetamido] penicillanic acid -Cyclohexanone (49' g) malonic acid (52 g), and ammonium acetate (77 g) were heated at reflux for six hours in 95% ethanol (150 ml.). The solution was allowed to cool, the crude product (47 g) collected by filtration and recrystallised from 95% ethanol to yield (l-aminocyclohexyl) acetic acid [(40 g) m.p. 240C: (found C, 61.0; H, 9.7; N, 8.9; C8H15N02 requires C, 61~
H, 9.6; N, 8.9%) n.m.r. ~ppm (D2O) 1.5-1.8 (b.s.,10~l), 2.5 (s, 2H),Ii.r. ~ ax (mull) 215d cm (s, -NH3( ) overtone), 1610 and 1495 cm (s,s, - N~13( ), 1560 cm 1 (b.s. -CO2( )~ .
The amino acid (39.25 g) was suspended in dry methylene di-chloride and dry hydrogen chloride gas passed through for 30 minutes. The white solid (l-amino-cyclohexyl) acetic acid hydrochloride ~(46.8 g) m.p. 200-204 C,: (found C, 49.6; H, 8.4; N, 7.2; Cl, 18.4; C8H16ClN02 requires C, 49-6;
Il, 8.3; N, 7.2; Cl, 18.3%~ was collected by filtra-tion, washed with dry methylene dichloride, dried in vacuo, and then added to a solution of thionyl chloride (27 ml.) in methylene dichloride (600 ml.). The suspension was heated gently at reflux for two hours to give a clear solution. The solvent was removed under reduced pressure, dry ether (500 ml.) was added, and evaporati.on was repeated under reduced pressure. The partial:l~ crystalline residue was triturated with dry ether and collected by ~iltration to give the acid chloride hydrochloride . .. , , . ,, . , . : .

.. . ..
.. ,: , ., : , ' ' , - ~ :
. . . : .

[(41 g), m.pl 117 C: (~ound C1, 33.0; C8~115C12NO requires Cl, 33.8%) i.r.
(mull) 1800 cm 1 (s, -CO.Cl)] .
Solu~ions o~ the acid chloride hydrochloride (23.3 g) in methyl-ene dichloride (250 ml.) and triethylamine (14.0 ml.) in methylene dichloride (150 ml.) were added concurrently over a period of 20 minutes to a solution of triethylammonium 6-amino penicillanate (31.7 g) in methylene dichloride (250 ml.) at room temperature. Stirring was continued for a further 30 minutes, when the precipitated pale yellow solid was filtered, well washed with methylene dichloride and dried in vacuo to yield 6-[(1--aminocyclohexyl) acetamido] -peniclllanate~ L(28.0 g), i.r. ~ (mull) 1775 cm (s./~-lactam C0) . This material, when sub~ected to paper chromatography in butanol:ethanol:water:
4:1:5 top layer, revealed a single zone of anti~acteria]. activity at an Rf value of 0.28 and was estimated by colorimetric assay with hydroxylamine to be 71% pure.
EXAMPLE III
Preparation of 6-r4-amino-1-thiacyclohexan-4-y1 acetamido~ penic:illanic acid l-thiacyclohexan-4-one. (17.4 g), was added to malonic acid (17.1 g), and ammonium acetate, (23.1 g), in ethyl alcohol. (300 ml.). This slurry was heated to give a solid mass which on ~urther heating gave a suspension.
Heating was continued for a ~urther 4 hours at reflux and then filtered hot to give (4-amino-1-thiacyclohex~4-yl) ace~ic acid,r(14.17g),`m.p. 232-235(d), found C. 47.6; H, 7.5; N, 8.0; S, 18.6; C7H13N02S requires C, 48.0; H, 7.5;
N, 8.0; S, 18.3/].
The amino acid, (10 g), was suspended in dry methylene dichloride, (200 ml.), and dry hydrogen chloride gas passed for 30 minutes. The white amino acid hydrochloride, (11.8 g), was collected ~y filtration, washed with methylene dichloride dried i.n ~acuo, and then added to a solution of thionyl chloride, __ (36 ml.), in methylene dichloride, (100 ml.), and gently refluxed for 30 minutes.

.
., . :, . .

.
: ' .

3~5~

The solvents were remoyed under reduced p~essll~e, dry ether, (20 ml.), was then added and removed under reduceci pressure twice. Finally dry ether was added and the crude acld chloride hydrochloride L(10.0 g), i.r. y (mull) 1795 cm 1 acid chl.oride carbonyl], collected by filtration and dried in vacuo over phosphorus pentoxide.
The crude acid chloride hydrochloride, (1.8 g), was added to a solution of triethylammonium 6-aminopencill'anate, (2.2`g), in methylene di-chloride, (25 ml.), concurrently with triethylamine, (0.7 g). The reaction mixture was stirred for I hour, filtered' and the collected solid slurried with isopropanol and re-filtered to give the penicillin, ~(1.05 g), i.r.~ (mull) 1775 cm 1 ~ lactam car~onyl~. When subjected to paper chromatography in ~utanol:ethanol:water, a single zone of antibacterial inhibition was obtained at an Rf of 0.24 and it was estimated by colorime-tric assay with hydroxylamine to be 40% pure.
EXAMPLE IV

reparation of 6- rDL-3-amino-3-(cyclohexane-1 yl) propionamido] penicillanic a_ Cyclohexane-l-aldehyde (56 g)~ malonic acid (52 g) and ammonium`
acetate (77 g) were heated at reflux!for 24 hours in 95% ethano]. (150 ml.).
The solution was allowed to cool, the crude amino acid~(41 g m.p. 219-220~(d)) collected by filtration, washed with ethanol and dried in vacuo over phosphorus pentoxide. Récrystallisation from 5N. hydrochloric acid ga~e white needles of of 3-amino-3-cyclohexyl propionic acid hydrochloride [(39 g) m.p. 214-215(d):
(found C, 52.1; H, 8.9; N, 6.9; CgH18ClN02 requires C, 52.2, H, 8.7; N, 6.7%) ' n.m.r. ~ppm. [(CD3)2SO] 8.3 (3H, m), 3.3 (H,m), 2.7 (2H,d), 2.0~1.0 (111l,-m), : i.r. Ymax (KBr) 3240 and 1600 (NH), 2600 (NH ), 1710 cm 1 (C0)~.
The amino acid hydrochloride (6.2 g) was suspended in dry ~ethylene dichloride (75 ml.) and dr~ hydrogen chloride gas passed for 30 minutes at 5. Phosphorus pentachloride (9.~15 g) was added slowly and then stirred for . .

:' , "':, ' ' :' . . :, , , : . .: . , . ', . ; ,: .. . ' ': . : ' ' ', ,: :. , . .: ': ' ' ..':' ,.'; . ' , ~., .: ... :... :. . : ...... ... . .. . . . ~.. , . . . :: . .

~5~ 7 one hour furthe~, with exclusion o~ moisture~ to giye a ~ ear solution.
~nhydrous acetone ~75 ml.) was then carefully added with stirring and evaporated to dryness under reduced pressure after 30 ~inutes. The white residue was triturated with anhydrous diethyl ether, filtered and dried in vacuo over phosphorus pentoxide to give acid chloride hydrochloride L(6.81 g) i.r. y x (mull) 2700 and 1~00 (Nll ), 1790 (C0~, 1600 cm (N~l)].
A vigorously stirred, finely divided suspension of 6-amino penicillanic acid (prepared by dissolving triethylammonium 6~amino penicillanate, (6.35 g) in 25% aqueous acetone at -5 and adjusting the pH to 2.5 with cold 5N hydrochloride acid) was treated portion wise with the acid chloride hydro-chloride (6.75 g) with the reaction mixture maintained at pH 2.5 to 3.5 by the careful add;Ltion of triethylamine. After 30 minutes when the addition was complete, a clear solution was obtained. Stirrin~ was continued for 20 minutes further at 0-5 when the pH was readjusted to 4.5 with triethylamine and the acetone removed under reduced pressure. The pH was adjusted to 7.0 with tri-etllylamine and the solution evaporated to dryness under reduced pressure. The resulting wllite solid was dried in vacuo over phosphorus pentoxide and then thoroughly triturated with dry methylene dichloride and filtered to give the required penicillin' ~(5.4 g), m.p. 170(d), n.m.r. ~p.p.m. t(CD3)2SO~ 5.5 (2H, s); 4.2 (H,s); 3.1 (H, t); 2.75 (2H, m), 2.0-1.0 17H~ m); i.r. ~max (mull) 3300 (NH), 1785 (lactam C0)~ 1660 cm 1 (amide C0)]. The producL, when sub~ected to paper chromatography in butanol:ethanol:water: 4:1:5, (top layer)9 revealed a single zone o inhlbition at an Rf value of 0.44 and was estimated by colori-metric assay with hydroxylamine to be 78% pure.
EXAMPLE V

Prepsr~tion Or 6~!DL 3-amino-3-(cyclollex-1, 4-dien-1-yl) propionamido~ peni-_llanic acid To a stirred solutio~l of DL-3-amino-3 phenyl propionic acid - :10 -. ~

~L~5~8~L79 propionic acid (16.3 g) in liquid a~monia (800 ml.), tetrahydrofur~n (200 ml.) and tertiary butanol (200 ml.) contained in a vacuum jacketed vessel, was added strips of lithium (8.0 g) over a period of ~ hours, when a permanent blue colour was obtained. ~mmonium chloride (62 g) was cautiously added and the ammonia allowed to slowly evaporate overnight. The white residue was dissolved in water (200 ml.), the volume reduced to about 50 ml. and the pH adjusted to 5.5. The white solid was collected and recrystallised from 5N. hydrochloric acid to give the corresponding propionic acid hydrochloride ~(15.0 g m.p. 210-212 : (found C, 52.8; H, 7.0; N, 6.ô; CgH14C1N02 requires C, 53.1; H, 6.9;
N, 6-8%) n m.r. op.p.m. [(CD3)SO~ 9.0 (3H, m), 5.85 (H, s); 5.7 (2H, s)- 3~.85 (H, t); 2-7 (6H, m) i.r.)fmaX (mull) 2600 and 1950 (NH~), 1730 (CO), 1600 cm~
(C = C)~.
The amino acid (6.1 g) was di.ssol~ed in 1.ON potassium hydroxide solution (60 ml.), the solution evaporated to dryness under reduced pressure and dried in vacuo over phosphorus pentoxide for 24 hours. The potassium salts was suspended in absolute ethanol (50 ml.), methylacetoacetate (3.48 g) added and the mixture gently refluxed for 2 hours. The clear solution was reduced to . . .
about 10 ml. and triturated with ether. The white crysta].Iinè solid was collected ~y filtration, washed with ether and dried ~n vacùo over phosphorus pentoxide to giye the corresponding potassium salt ~(9!0 g) m.p. 234-5, :
(found C, 55.6; H, 6.1; N, 4.3; C14H18KN04 re~uires C, 55.5; H, 5.9; N, 4.4%).
n-m-r- Op.p.m. r(CD3)2SO] 5.67 (H, s); 5.5 (2H, s); 4.25 (H, s); 3.85 (H, m), 3.5 (3H, s), 2.6 (6H, m); 1.85 (3H, s), i.r.-yma (K~r) 1640 (C03, 1600 (C = C), 1580 cm 1 (CO).
The potassium salt of the N-protected amino acid (3.5g) was suspended in dry acetone (30 ml.), chilled to -10 and treated with ethyl chloroformate (1.08 g) and one microdrop of N-methyl morpholine with stirring.
The mixture was maintained at -10 ~or 30 minutes, chilled to -30~ filtered - 11 - ' .. . , ~ . : . . . . , . . ... .: , .
, ': . " .. . ' ' .. :. ' ,. : '' . ' ..

: . .; . . . . . .
.. , . , , ~ .. . .. . ...

~L~56~L7 th~ough kieslguhr and added to a vigorously stirred solution of triethylammonium 6-aminopenicillanate (3.16 g) in 50% aqueous acetone (60 ml.) at 0. After stirring for 1 hour with no further external cooling, ~he acetone was removed under reduced pressure, the concentrate co~ered with ethyl ace-tate (30 ml.) and adjusted to pl-l 1.5, with stirring, by the addition of concentrated hydrochloric acid at 5 for 20 minutes. After separation o~ the phases, the aqueous layer was readjusted to pH 6.0 with triethylamine and evaporated to dryness under reduced pressure. The white residue was dried over phosphorus pentoxide for 24 hours, thoroughly diges~ed with methylene dichloride (200 ml.), filtered to gi.ve the penicillin, [(1.8 g) m.p., 165 (d), n.m.r. ~p.p.m. L(CD3)2SO] 6.5 (3H, s); 5.8 (3H, s); 5.35 (2H, s); 4.05 {H, s) 3.85 (H, tj; 2.75 (6H, m);
1.5 (611, d) i.r. y a (mull) 340d (HN); 1770 (lactam C0), 1665 (amide C0), 1580 (Co)3 .
The material, when subjected to paper chromatography revealed a single zone of inhibition at-Rf 0.26 and was estimated to be 75% pure by colorimetric assay with hydroxylamine. _ EXAMPLE VI

Preparation of 6-rDL-3-amino-3~(cyclohex-3-en~ yl) propionamido~penicillanic acid DL-3-amino-3-cyclohex-3-en-yl propionic acid hydrochloride, [(42 g) m.p. 197-198 : (found C, 52.0; H, 7.7, N, 7.0; CgH16ClN02 requires C, 52.2; H, 7.7, N, 6.8%) n.m.r. (CD3)2So3 ~p.p.m. 8.3 (3H, m); 6.6 ~2H, s), 3.35 (H, m); 2.75 (211, d); 2.0 (7H, m) i.r. ~max (mull) 2600 and 1940 (NH ), 1710 (C0) 1600 cm 1 (C = C)3, was prepared exactly as described in Example (IV) when cyclohexane-1-aldehyde was replaced by cyclohex-3-ene-1-aldehyde (55 g).
The amino acid hydrochloride (9.0 g) was added to a.solution of sodium ethoxide ~prepared :erom sodlum (2il g) and absolute ethanol (100 ml.)3 in ethanol. Methylacetoacetate (5.5 g) was then added, the mixture refluxed ~or 16 hours and ~iltered.` The ~iltrate was concentrated under reduced pressure 12 -- :

~56~

and triturated with dry diethyl ether. The white solid was collected, washed with dry ether and dried _ ~acuo over phosphorus pentoxide at 60 for 2~1 hours,to give the corres~onding sodium salt [(9.0 g) m.p. 114-115 : (found C, 53.2;
H, 6.9; N, 4.3; C1~1120NaN04 H20 requires C, 54.8; H, 7.1; N, 4.5%) n.m.r, ~p.p.m.
L(CD3)SO] 4.0 (~I, d); 5.78 (211, s); 4.25 (Il, s) 3.6 ~Fl, m); 3.5 (311 S);

2.2 (2H, m); 1.9 (11~1, m), i.r. ~ (K~r) 1640 (CO), 1580 cm (C2 )' The penicillin, [(1.1 g) m.p. 160 (d), n.m.r. ~p.p.m. ~(CD3)2 S0 -~ D20] 5.7 (2H, s); 5.4 (2H, s); 4.1 (H, s); 3.5 ~H, t); 2.7 (2H, m);
2.0-1.5 (13~1, m), i.r. ~ (KBr) 1760 (lactam C0), 1650 cm (amide CO)] was prepared and isolated exactly as described in the latter part of Example V
when the N-protected amino acid (2.8 g) was utilised. This material, upon paper chromatography revealed a single zone of Rf 0.43 and was estimated by colorimetric assay with hydroxylamine to be 84% pure.
~XA~LE VII
Preparation of 6-~DL-2-amino (cyclohex-l-en-l-yl) propionamido~ penicillanic acid

3-amino-3-cyclohex-1, 4-dienyl propionic acid hydrochloride (6.2 g), dissolved in 5N hydrochloric acid (100 ml.) was hydrogenated under slight positive pressure at room temperature with prehydrogenated 5% palladium on carbon ~15 g). After 40 minutes, when the rate of hydrogenation had lessened the r~action was stopped filtered through kieslguhr and evaporated to dryness under reduced pressure. The white solid, 3-amino-3-cyclohex-1-enyl propionic acid [~6.0g? m.p. 162-163 nbm.r. p.p.m. [(CD3)2SO] 5.9 (H, s); 3-85 (H, tj;
.
2.75 (2H, d); 2.0-1.5 (8H, m) i.r. Ymax (mull) 2600 and 1940 (NH), 1820 (CO)~
1600 cm 1 (C ~ C)~ was identified as its hydrochloride.
`The sodium salt [~10.1 g) m.p. 124-126 : (found C, 54.8;
Il, 7.2; N, 4.5; C]4H20NaN04.H20 requires C, 54.8, H, 8.1, N, 4.5%) n.m.r.
[(CD3)2S0] Sp.p.m. 8.9 (H, d); 5.5 ~H, ~i); 4.3 (H, s); 3.7 (H, mj; 3.5 (3H, s);
2,2 (2H, m); 2.0-1.5 (llH, m), ;L.r. ~ (mull) 1640 (C0), 1580 cm (C02 )]

~s~

was prepared as described in lixall1ple ~7I except the product crysLallised out upon cooling of the reaction mixture.
The penicillin, L(1.6 g) m.p. 170 (d), n.m~r. ~p.p.m. [(CD3)2SO
D20] 5.8 (E-1, s); 5.~1 (211, s); 4.6 (H, s); 3.8 (H, m); 2.8 ~2H, m); 2.0-l.5 (1411, m), i.r. y (mull) 1770 (lactam C0), 1660 (amide CO), 1590 cm 1 C02 )], was prepared and isolated exactly as detailed in Example VI. The product, when subjected to paper chromatography indicated a single ~one of inhibition at RE oE 0.42 and was estimated to be 567~ pure by colorimetric assay with hydroxylamine.
E~rPLI~ ~IIII
6~ ~DL-3-amino~3-methyl butyramido~ penicillanic acid 3-amino-3-methyl butyric acid (3.0 g) was suspended in dry methylene dichloride (50 ml.) and dry hydrogen chloride passed for 25 minutes at 0-5C. Phosphorus pentachloride (6.9 g) was added portion wise over 5 minutes with continuous stirring and the exclusion of moisture. A pale yellow solution was quickly obtained. ~fter 1.5 hours, dry acetone (5 ml.) was added and after 30 minutes the reaction mixture was evaporated to dryness under reduced pressure. Dry toluene (3 x 10 ml.) was added and re-evaporated under reduced pressure. The resultant white solid was triturated with dry diethyl ether, collected by filtration in a dry box and after drying ln vacuo over phosphorus pentoxide gave the acid chloride hydrochloride ~i.r. y a (mull) -2070 (NH+), 1780 cm 1 (CQj]
~ cooled suspension of the acid chloride hydrochloride in methylene dichloride (70 ml.) and methylamine ~3.5 ml.) were added concurrently over 5 minutes to an ice-cold solution of triethylammonium 6-amino penicillanate (8.13 g) in dry methylene chloride (35 ml.). After stirring for 45 minutes, the white solid was collected by fi:l t~ation? well washed with methylene di-chloride and dried in racuo over phosphorus pentox.ide to give the penicillin .

, i " . . . , ,, : :. . . ..

~s~

L~7.0 g) n-m-r- Sp~p-m~ (P20 ~ Na~lC03~ 6.55 (2H, m); 4.25 (H, s); 2.7 ~2H, 9);
1.63-153 (611, s); 1.45 (6H, s)` i.r. y (mull) 1770 cm (lactam CO)] . This material, when subjected to paper chromatography indicated an R~ value of 0.16 and was estimated by colorimetrlc assay with hydroxylamlne to be 67% pure.
~XAMPLE IX

Preliminary in vitro testing of 6-¦(l-amlno cyclohexyl) acetamido~ penlclllanic acld (XI):

f \ / CH3 ~ CH2CO.NH.CH CH C--CH3 CO N - CH.COOH
NH2 (XI) show it to have the same type of antibacteria:L spectrum as ampicillin. Although it was slightly less active than ampicillin against most strains of bacteria, it was considerably more active than cyclocil:lin (IV). Moreover, 6- [(l-amino-cyclohexyl) acetamido] penicillanic acid (XI) gave appreciably better blood levels than ampicillin when both compounds were given by mouth at 100 mg./kg.
to squirrel monkeys-AVER~GE SERUM C NCNS. ~ug/ml) Z0 ~ hr. 1 hr. 1 2 hr. 4 hr. 6 hr.
. .
Compound (XI~ 14.65 l9.9 13.7 4.6 0.87 mpicillin 6.5 11.1 4.0 1.2 0.4 EX~kPLE X
Preliminary blood level studies of 6-[DL-2-amino-3-methyl butyl carbamido~ -penicillanlc acld (X) when given by mouth at 100mg/kg to squirrel monkeys showed tliat higher and more prolonged blood levels could be achieved than with ampicillin.
~ :

13 \ / \ / 3 C11 - Cli.CH .CO.NH.CH --Cll C CH

NH2CO -- N - CH.CO2H, (X) Results A~ERAGE SERU~I CONCNS. (u~/ml) hr.1 hr. 2 hr. 4 hr. ¦ 6 hr.
, Compound X 16.832.1 28.0 20.3 4.8 -Alnpicillin 10.1 8.1 4.7 2.1 0.45 EXAMPLE XI
The following Table records the blood levels achieved when two more of the compounds of this invention were given by mouth at 100 mg/kg to squirrel monkeys. The results achieved with ampicillin are included for comparison purposes.
:
R1 / ~\ / CH3 ~ C-- CH ---C0-- NH -- CH ---CH C - - CH
IH2 2 ~ IV) CO N-- - CH -COOH

- ~6 -.'": .` '.: ' ' ' ~ .''': ' ' `

~t~5~

~ Mean co~c.~lg/ml at hours a~ter dosing Compound 0.5 1.0 7.. 0 4'0 ¦ 6-0 . _ (IV): R= ~

R1 H12.2 18.7 15.9 5.9 2.0 (IV): R= ~ ~ -R1 = H 5.8 7.1 6.1 2.6 j 1.3 Ampicillin6.6 12.9 5.0 1.9 ¦ 0.8 These results when taken together with the results recorded in E~amples (IX) a~d (X) show that the compounds of this invention are generally better absorbed and/or give more prolonged blood levels in squirrel monkeys than ampicill:in. In addition, from these results it appears that compounds of formula (IV) where R and Rl contain no unsaturation are bétter absorbed than compounds where R or R is an unsaturated group.

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~: . . , ' . , : . : , .

Claims (20)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE
IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of penicillins of formula (IV) (IV) and non-toxic salts and hydrolyzable esters thereof, in which formula R is a C1 to C6 straight or branched chain alkyl group, a C5 to C7 cycloalkyl or cycloalkenyl group; R1 is hydrogen or a C1 to C6 alkyl group or R and R1 taken together represent a C5 alkylene group which may be interrupted by a sulphur atom, in which process 6-aminopenicillanic acid or a salt or ester thereof or the reaction product of 6-aminopenicillanic acid with an organo silicon compound is treated with a reactive acylating derivative of an acid of the general formula (V) (V) wherein R and R1 are as defined above and X is a nitrogen containing group which may be converted into a primary amino group, and subsequently convertingthe group X into a primary amino group and when required converting the resulting compounds into their non-toxic salts or hydrolyzable esters.

2. A process as claimed in claim 1 wherein the reactive derivative of the acid of formula (V) is the acid halide, azide, anhydride, mixed anhydride or the reactive intermediate formed from the acid and a carbodiimide or carbonyldiimidazole.

3. A process as claimed in claim 1 or claim 2 wherein the group X is a protonated amino, azide, benzyloxycarbonylamino or substituted benzyloxy-carbonylamino group or a group of formula VII or VIII

(VII) (VIII) wherein R2 is a C1 ta C6 alkyl group, an aryl group or C1 to C6 alkoxy group, R3 is hydrogen, R4 is C1-6 alkoxy, and Z represents the residue of a substituted or unsubstituted benzene or naphthalene ring.

4. A process for preparing a compound of the formula which comprises reacting 6-aminopenicillanic acid, or a functional derivative thereof with an acid of the formula or a functional derivative thereof, wherein Z is a primary amino group or a N-containing group convertible to a primary amino group and when required converting the reeulting compound into a non-toxic salt or hydrolyzable ester.

5. A compound of formula or a non-toxic salt or hydrolyzable ester thereof whenever prepared by the process of claim 4 or by its obvious chemical equivalent.

6. The process of claim 1 wherein the acid of the general formula (V) is 3-amino-4-methyl pentanoic acid chloride-hydrochloride.

7. 6-[DL-3-amino-4-methyl pentanyl amido]-penicillanic acid when prepared by the process of claim 6 or by its obvious chemical equivalent.

8. The process of claim 1 wherein the acid of the general formula (V) is (4-amino-1-thiacyclohex-4-yl) acetic acid chloride-hydrochloride.

9. 6-[4-amino-1-thiacyclohexan-4-yl acetamido]penicillanic acid when prepared by the process of claim 8 or by its obvious chemical equivalent.

10. The process of claim 1 wherein the acid of the general formula (V) is 3-amino 3-cyclohexyl propionic acid chloride-hydrochloride.

11. 6-[DL-3-amino-3- (cyclohexan-1-yl) propionamido]penicillanic acid when prepared by the process of claim 10 or by its obvious chemical equivalent.

12. Ihe process of claim 1 wherein the acid of the general formula (V) is 3-(N-1-methoxycarbonylpropen-2-yl)amino-3-cyclohexa-1, 4-dienyl propionic acid,potassium salt.

13. 6-[DL-3-amino-3-(cyclohex-1, 4-dien-1-yl)propionamido]penicillanic acid when prepared by the process of claim 12 or by its obvious chemical equivalent.

14. The process of claim 1 wherein the acid of the general formula (V) is 3-(N-1-methoxycarbonylpropen-2-yl)amino-3-cyclohex-3-en-yl propionic acid, sodium salt.

15. 6-[DL-3-amino-3-(cyclohex-3-en-l-yl) propionamido]penicillanic acid when prepared by the process of claim 14 or by its obvious chemical equivalent.

16. The process of claim 1 wherein the acid of the general formula (V) is 3-amino-3-cyclohex-1-enyl propionic acid, sodium salt.

17. 6-[DL-3-amino-(cyclohex-1-en-1-yl) propionamid]penicillanic acid when prepared by the process of claim 16 or by its obvious chemical equivalent.

18. The process of claim 1 wherein the acid of the general formula (V) is 3-amino-3-methyl butyric acid chloride-hydrochloride.

19. 6-[DL-3-amino-3-methyl butyramido]penicillanic acid when prepared by the process of claim 18 or by its obvious chemical equivalent.

20. Penicillins of formula (IV) as defined in claim 1 and non-toxic salts and hydrolyzable esters thereof whenever prepared by the process of claim 1 or by its obvious chemical equivalent.